PROJECT SUMMARY Infection by the unicellular parasite Giardia lamblia leads to intestinal distress and can cause severe malnutrition in children who remain untreated. Giardia infects over 300 million people annually, with 20% of those cases being resistant to front-line treatment. Giardia attaches to the intestinal epithelium of its hosts with two cellular structures: the ventral disk and the ventrolateral flange. Attachment research over the past fifty years has focused on the suction-cup like ventral disk, whereas the lamellipodium-like ventrolateral flange remains biochemically and structurally uncharacterized. Published work and our preliminary data indicate that the cytoskeletal protein actin plays a central role in the ventrolateral flange. Giardia has the most divergent actin sequence identified in eukaryotes and lacks all of the canonical, eukaryotic actin-binding proteins, yet actin is involved in several core cellular processes in Giardia, including cytokinesis and cell morphogenesis. Together this suggests that the mechanisms by which Giardia builds actin-based cellular structures differ significantly from other eukaryotes and that Giardia likely contains non-canonical regulators and novel actin-binding proteins that modulate the actin cytoskeleton. This proposal describes approaches to characterize both actin (Aim 1) and the flange (Aim 2) from Giardia using cryo-electron microscopy and super-resolution imaging. This work will define the molecular underpinnings of the flange ultrastructure (Aim 2) and reveal the conserved and divergent mechanisms of actin assembly, dynamics, and regulation in Giardia (Aim 1). Identifying functional properties that distinguish the actin cytoskeleton of Giardia from its vertebrate homolog will provide opportunities for new drugs that specifically target the parasite without harming the host. This work will also generate fundamental insights into core actin properties. This interdisciplinary research project is an exceptional postdoctoral training opportunity, leveraging questions that sit at the nexus of several scientific fields and the resources available at the University of Washington into an impactful research strategy and training plan. The PI will work jointly with two faculty mentors: Dr. Justin Kollman, an expert in cryo-electron microscopy and divergent actins; and Dr. Alex Paredez, a leader in research on Giardia who has developed many of the tools used to study actin in the parasite. In addition, the PI has assembled a team of collaborators and mentors at the University and around the country. The University will provide extensive training in the responsible conduct of research, professional skills, and teaching pedagogies. Though the execution of the research strategy and training plan, the PI will gain technical expertise in cryo-electron microscopy and super-resolution microscopy as well as the key career skills necessary to launch an academic research laboratory. Together, this research and training will prepare the PI to conduct studies bridging the resolution gap between individual proteins and the cell-level interactions of hosts and pathogens.